Mono(2-ethylhexyl) phthalate induces autophagy-dependent apoptosis through lysosomal-mitochondrial axis in human endothelial cells

Accepted Manuscript Mono(2-ethylhexyl) phthalate induces autophagy-dependent apoptosis through lysosomal-mitochondrial axis in human endothelial cells Xueyan Wu, Liping Jiang, Xiance Sun, Xiaofeng Yao, Yueran Bai, Xiaofang Liu, Nairong Liu, Xingyue Zhai, Shaopeng Wang, Guang Yang PII:

S0278-6915(17)30305-8

DOI:

10.1016/j.fct.2017.05.069

Reference:

FCT 9104

To appear in:

Food and Chemical Toxicology

Received Date: 21 January 2017 Revised Date:

5 May 2017

Accepted Date: 30 May 2017

Please cite this article as: Wu, X., Jiang, L., Sun, X., Yao, X., Bai, Y., Liu, X., Liu, N., Zhai, X., Wang, S., Yang, G., Mono(2-ethylhexyl) phthalate induces autophagy-dependent apoptosis through lysosomalmitochondrial axis in human endothelial cells, Food and Chemical Toxicology (2017), doi: 10.1016/ j.fct.2017.05.069. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Mono(2-ethylhexyl) phthalate Induces Autophagy-Dependent Apoptosis through Lysosomal-Mitochondrial Axis in Human

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Endothelial Cells

Xueyan Wu1, Liping Jiang2, Xiance Sun2, Xiaofeng Yao2, Yueran Bai1, Xiaofang Liu1,

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Nairong Liu1, Xingyue Zhai1, Shaopeng Wang3*, Guang Yang1*

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Department of Food Nutrition and Safety, Dalian Medical University, Dalian 116044, China

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Liaoning Anti-degenerative Diseases Natural Products Engineering Technology Research

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Center, Dalian Medical University, Dalian 116044, China

Department of Cardiology, the First Affiliated Hospital of Dalian Medical University,

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No. 222. Zhongshan Road, Dalian 116011, China

* Corresponding authors. E-mail addresses: [email protected] (Guang Yang), [email protected] (Shaopeng Wang).

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1. Introduction Di(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer contained within the manufacturing of polyvinyl chloride (PVC) consumer, plastic bags, industrial paints,

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cosmetics, food packaging as well as blood transfusion packs (Muczynski et al., 2012). Additionally, the global production volume of DEHP is of large quantity, approximately 3 million tonnes a year. This is of special concern because the exposure to DEHP is

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widespread and frequent. Phthalate-containing plastics can leach phthalates readily into bodily fluids; consequently, food containers and medical devices such as blood bags and

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intravenous tubing can pose a significant route of human exposure to DEHP (Benjamin Moyer, Hixon, 2012). Mono(2-ethylhexyl) phthalate (MEHP) is the bioactive monoester metabolite of DEHP and excreted in the urine (Mcmaster et al., 2010). Data collected from the NHANES cohort between 1999 to 2006 show measurable levels of MEHP in

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80% of urine samples analyzed (Kelly K. Ferguson, Rita Loch-Caruso, 2011). Significant

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from a toxicological standpoint, it is hypothesized that the toxicity of DEHP is mediated by MEHP in many organ systems (Hannon et al., 2015).

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The toxic effects of MEHP have been described partially in animal models and tissue culture. It has been proved that MEHP exposure altered reproductive function by

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affecting testicular function in hamsters, caused alteration of thyroid hormone levels in men, and induced inflammatory state by activated the adaptive immune system(Parks et al., 2000; Meeker et al., 2007; Campioli et al., 2014). Moreover, effects on the hepatic, urologic systems, carcinogenicity, developmental toxicities have been reported (Rosado-Berrios et al., 2011). However, reports about the cardiovascular toxicity of MEHP are rare, remains largely unknown.

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Apoptosis, a tightly regulated cell deletion process, plays an essential role in various cardiovascular diseases (Kim and Kang, 2010). Autophagy and apoptosis command the turnover of organelles and proteins within cells. In general terms, autophagy suppresses

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apoptosis, and apoptosis-associated caspase activation shuts off the autophagic process. However, in special cases, autophagy may contribute to apoptotic cell death through unchecked degradative processes (Mariño et al., 2014). Autophagy and apoptosis often

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occur in the same cell, mostly in a sequence in which autophagy precedes apoptosis (Maiuri et al., 2007). Thus, an investigation is needed to understand how the entire

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molecular machinery of apoptosis and autophagy are coordinate to influence the cardiovascular cell fate, and to examine if any possible relationship existed, in MEHP-treated cells.

Since MEHP has been shown to induce apoptosis in some cells, fully understanding

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the regulatory mechanisms of apoptotic signaling is crucial (Meruvu et al., 2016). Many

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signal transduction pathways elicited by cell-intrinsic stress modulate both autophagy and apoptosis (Mariño et al., 2014). It is indicated that the cytoplasmic pool of p53, a tumor

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suppressor protein functioned as a signal transduction integrator, blocks autophagy, while the nuclear translocation of p53 can stimulate autophagy (Tasdemir et al., 2008).

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Moreover, p53 can directly promote mitochondrial outer membrane permeabilization (MOMP), then MOMP leads to the release of different apoptosis-mediating molecules, for instance, cytochrome c, which activates caspase 9 and successively cleaves caspase 3 and caspase 7, thereby setting off the apoptotic cascade (Nikoletopoulou et al., 2013). In additionally, BH3-only proteins as well as Ser/Thr kinases, including AKT, JNK and DAPK have also been linked to both autophagy and apoptosis (Mariño et al., 2014).

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MOMP was occurred downstream of lysosomal membrane permeabilization (LMP), and the discharge of the lysosomal protease cathepsins into the cytosol leads to apoptosis (Eno et al., 2013).

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Overall, our study offers comprehensive insights into the effects of MEHP treatment on LMP, the role of LMP in MEHP-induced apoptosis, and the relationship between autophagy and apoptosis in MEHP-treated cells.

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2. Materials and methods 2.1. Cell culture and treatment

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EA.hy 926 cells were obtained from Peking Union Medical College, and cultured in DMEM medium (Gibco) containing 10% fetal bovine serum (Biological Industries), penicillin (100 U/mL) and streptomycin (100 g/mL) at 37 °C in a humidified atmosphere of 5% CO2. MEHP was obtained from Sigma Aldrich (CAS No. 4376-20-9, assay: 97%),

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and dissolved in dimethyl sulfoxide (DMSO, Sigma, assay≥99.5%) to prepare a stock

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solution of 100 mM. EA.hy 926 cells were treated with 200 μM MEHP. In inhibition experiments, cells were pretreated with different inhibitors: 1 mM 3MA (Sigma, assay:

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98%), 10 μM CA-074 Me (AdooQ, assay>98%) for 2 h before the exposure to MEHP for 12 h. The DMSO concentration was 0.2% for all exposure groups and solvent controls.

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2.2. Cell viability assays

The cytotoxicity of MEHP was detected by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. EA.hy 926 Cells were seeded in 96-well plates at a density of 5 × 104 cells per well, treated with medium alone, DMSO (solvent control), or MEHP (25, 50, 100, 200, 400, 800 μM) for 24 h. After treatment, DMEM with MTT reagent (5 mg/ml in sterile PBS) was added and incubated for 4 h at 37 °C.

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The medium was discarded thereafter, and DMSO was supplemented. The absorbance was measured at 570 nm using a Bio-Rad Microplate Reader and the cell viability (%) was calculated as (A570 of treated group/A570 of negative control) ×100.

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2.3. Western blot analysis

The EA.hy 926 cells were harvested, and lysates were prepared using lysis buffer provided with a protein extraction kit (Keygen Biotech). To assess subcellular

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relocalization of cathepsin B and cytochrome c, immunoblotting was conducted using cytosolic extracts, cells were washed twice with ice-cold PBS and then with 0.4 mL of

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permeabilization buffer [20 mM HEPES (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 250 mM sucrose, 1 mM DTT, 1.5 mM phenylmethylsulfonyl fluoride, 3 μg/mL leupeptin, and 20 μg/mL aprotinin]. Protein concentration was determined by the BCA method. Samples were resolved by SDS-PAGE gels of appropriate percentage and

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transferred onto a polyvinylidene fluoride membrane. After blocking with 10% nonfat

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milk, the membranes were probed with the primary antibodies against LC3 (Sigma, CAT No. L7543), cathepsin B (Cell Signaling Technology, CAT No. #3178), cytochrome c

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(Proteintech, CAT No. 10993-1-AP), caspase 3 (Cell Signaling Technology, CAT No. #9662), and the internal control β-actin (Cell Signaling Technology, CAT No. #3700).

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Then, the membranes were incubated with anti-mouse or anti-rabbit HRP-conjugated secondary antibodies, and the bound antibody was visualized using the SuperSignal West Pico Kit (Thermo Scientific) and Bio-Rad ChemiDoc™ MP imaging system (Bio-Rad Laboraturies). Relative abundance was measured with Gel-Pro Analyzer 4.0 software. Experiments were repeated at least twice. 2.4. Lysosomal membrane stability assessment

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The stability of the lysosomal membrane was evaluated by the acridine orange (AO) relocation test. AO is a metachromatic fluorophore and a lysosomotropic base. In intact lysosome, accumulation of oligomeric protonated AO causes high concentrations of red

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fluorescence, while diffusion of lysosomal contents into the cytosol associated with LMP leads to the formation of monomeric deprotonated form of AO showing green fluorescence (Koshkaryev et al., 2012). Briefly, after different treatments, cells were

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harvested, stained with 1 μg/ml AO (Amresco) for 15 min at 37 °C in the dark. Samples were immediately observed and photographed using a fluorescence microscope

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(Olympus BX63). The stability of the lysosomal membrane was estimated by red fluorescence, applying Image-Pro Plus 6.0 software (Media Cybernetics). 2.5. ΔΨm (Mitochondrial membrane potential) assessment

ΔΨm of MEHP-treated EA.hy 926 cells was determined using the potentiometric

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fluorescent dye JC-1. JC-1 shows a shift from green fluorescence, which corresponded to

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depolarized/low ΔΨm (JC-1 monomers), to red fluorescence, which corresponded to polarized/normal ΔΨm (Jc-1 aggregates). In brief, after different treatments, cells were

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harvested, stained with 5 g/mL JC-1 (Beyotime Institute of Biotechnology) for 20 min at 37 °C in the dark. Samples were immediately observed and photographed using a

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fluorescence microscope (Olympus BX63), and for imaging of JC-1 monomers, a FITC filter was set, for imaging of JC-1 aggregates, a Cy3 filter was set. ΔΨm was estimated by the ratio of red/green fluorescence intensity (Kim et al., 2015), applying Image-Pro Plus 6.0 software (Media Cybernetics). 2.6. TUNEL assay Apoptosis was detected using In situ apoptosis detection kit (Keygen Biotech),

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following the manufacturer’s protocols. After TUNEL labeling, nucleus was counter stained with DAPI (Keygen Biotech). Briefly, after different treatments, cells were harvested, fixed with 4% paraformaldehyde and permeabilized in 0.1% Triton X-100.

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Then, TUNEL reagents were applied. After washing with PBS, the samples were mounted with DAPI. Fluorescent images were captured using a fluorescence microscope (Olympus BX63), and images for TUNEL stained cells and DAPI labeled nuclei were

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captured on five randomly chosen fields for each section. The number of TUNEL (+) cells was quantified by using Image-Pro Plus 6.0 software (Media Cybernetics).

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2.7. Statistical analysis

The values were presented as means ± standard deviation (SD), experiments were repeated at least twice, and analyzed using the SPSS 13.0 statistical software (IBM SPSS Software). Statistical analysis was performed using a one-way analysis of variance

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(ANOVA) followed by Student-Newman-Keuls (SNK) test, and P < 0.05 was considered

3. Results

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statistically significant.

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3.1.MEHP induced cell death in EA.hy926 cells In this study, MEHP caused a dose-dependent decrease in cell viability detected by

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the MTT assay (Figure 1). Treatment of EA.hy 926 cells with 100 μM, but not lower concentrations, significantly decreased cell viability, whereas 100 μM MEHP had no effect on apoptosis at 24 h (data not shown). Therefore, based on some references as well as our results, it is better to select 200 μM MEHP for our investigation (Tetz et al., 2013； Rosado-Berrios et al., 2011; Vetrano et al., 2010). We observed no differences in cytotoxicity with medium alone compared with 0.05% DMSO (solvent control).

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3.2. MEHP Induced LC3-Ⅱ Formation from the Early Period of Treatment LC3-II is the lipidated form of LC3, and has been widely used to study autophagy (Wang et al., 2015). Our result showed that the LC3-Ⅱexpression was upregulated after

significant in 3 h compared with the control (Figure 2). 3.3. LMP Induced by MEHP Was Autophagy Dependent

While the change was not

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treatment with 200 μM MEHP for 6 h, 12 h and 24 h,

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The stability of the lysosomal membrane in MEHP-treated cells was estimated by

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acridine orange (AO) staining. As illustrated in Figure 3A and B, exposed to 200 μM MEHP led to a decrease in the percentage of cells with red fluorescence (intact lysosome) in 12 h and 24 h compared with the control, indicative of LMP. However, no such effect of AO fluorescence indensity was observed in 6 h. In conjunction with the proof of LC3Ⅱexpression was upregulated early, these facts indicated that MEHP induced autophagy

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preceded LMP. To determine the role of autophagy in MEHP-induced LMP, EA.hy 926

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cells were pretreated with the autophagy inhibitor 3-methyladenine (3MA) at a concentration of 1 mM for 2 h. Pretreatment with 1 mM 3MA for 2 h attenuated

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MEHP-induced LMP in EA.hy 926 cells (Figure 3C and D). Thus, collectively these facts

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suggested that LMP induced by MEHP was autophagy dependent. 3.4. Effect of 3MA on the Expression of Cathepsin B Lysosomal release of cathepsin B can initiate or propagate pro-apoptotic signals (Bhoopathi et al., 2010). Western blot analysis pointed that the cathepsin B expression was significantly increased with 200 μM MEHP for 12 h and 24 h, while cells treated with MEHP for 6 h did not show appreciable difference in cathepsin B level as compared to control (Figure 4A and B). This event was corresponding to the data of MEHP-induced

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LMP. In addition, pretreated with 1 mM 3MA for 2 h attenuated MEHP-induced lysosomal release of cathepsin B in EA.hy 926 cells (Figure 4C and D). 3.5. Collapse of ΔΨm Induced by MEHP Was Autophagy Dependent

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Mitochondrial membrane degradation induced by cathepsin B can lead to apoptosis (Bossy-Wetzel et al., 1998). The state of the ΔΨm was assessed by the membrane-permeative potentiometric dye JC-1. The ΔΨm of cells was not altered in 6 h

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but obviously deceased in 12 h and 24 h after treated with 200 μM MEHP (Figure 5A and B). Additionally, pretreated with 1 mM 3MA for 2 h attenuated MEHP-induced collapse

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of ΔΨm in EA.hy 926 cells (Figure 5C and D).

3.6. Effect of 3MA on the Expression of Cytochrome c

Cytochrome c, a pro-apoptotic protein, released from mitochondria into the cytosol, is responsible for the dismantling of cells during apoptosis (Rehklau et al., 2012). Therefore,

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we assayed cytochrome c in cytosolic fractions by Western blot. Cytochrome c level was

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not significantly different from control at 6 h, in contrast, it was present a high abundance after treated with 200 μM MEHP for 12 h and 24 h (Figure 6A and B). Moreover,

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pretreated with 1 mM 3MA for 2 h attenuated MEHP-induced mitochondria release of cytochrome c in EA.hy 926 cells (Figure 6C and D).

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3.7. Apoptosis Induced by MEHP Was Autophagy Dependent TUNEL staining was performed to evaluate cellular apoptosis. We found elevated apoptosis in 12 h and 24 h after treated with 200 μM MEHP (Figure 7A and B). Furthermore, we analyzed the presence of the activated cleaved form of caspase 3 by immunoblot as a marker of apoptosis. As anticipated, the level of the cleaved caspase 3 was robustly increased when cells were exposed to 200 μM MEHP for 12 h and 24 h,

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while the change was not significant in 6 h compared with the control (Figure 7C and D). Besides, pretreated with 1 mM 3MA for 2 h attenuated MEHP-induced apoptosis in EA.hy 926 cells (Figure 6E and F), suggesting that the apoptosis induced by MEHP was

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autophagy dependent.

3.8. Effects of CA-074 Me on the Release of Cytochrome c and Apoptosis

To ascertain the role of cathepsin B in MEHP-induced mitochondriotoxicity and

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apoptosis, EA.hy 926 cells were pretreated with CA-074 Me, a cell-permeable-specific cathepsin B inhibitor, at a dose of 10 μM for 2 h. Interestingly, decreased mitochondria

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cytochrome c was readily detectable by immunoblotting in the cytosol of MEHP-treated cells(Figure 8A and B). Consistently, CA-074 Me significantly reduced apoptosis from MEHP (Figure 8C and D). These facts suggested that the lysosomal-mitochondrial axis may play a constructive role in MEHP-induced apoptosis.

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4. Discussion

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MEHP belongs to the phthalates family which was widely used in manufacture, and the accumulation of MEHP is of concern because MEHP is 20 times more toxic than

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DEHP in rats (Rael et al., 2009). However, the effects of MEHP upon human health continue to present controversy because relatively little is known about the effects on the system

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cardiovascular

at

a

cellular

level.

MEHP can

activate

peroxisome

proliferator-activated receptor-α (PPARα), and after forming heterodimers with a retinoid X receptor, the complex binds to a peroxisome proliferator response element, and activates the target genes associated with inflammation, metabolism of bile acid, lipoprotein, glucose and amino acid, fatty acid oxidation and apoptosis ( Lehraiki et al., 2009; Hayashi et al., 2011). Therefore MEHP may triggers apoptosis in EA.hy 926 cells.

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In this study, the pro-apoptotic activity of MEHP is well established, in the human umbilical vein endothelial cells, and the induction of autophagy was detected considering the increase of LC3-Ⅱlevel from the early stage of treatment. Since the interplay

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between autophagy and apoptosis is a matter of controversy, it aroused our interest in investigating the relationship of autophagy and apoptosis in MEHP-treated EA.hy 926 cells.

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In the current study, our results demonstrated that MEHP induced autophagy from 6 h, while the level of apoptosis increased from 12 h as well as the LMP and MOMP. We

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therefore hypothesized that there would exist a signaling pathway regulating both autophagic and apoptotic machinery, and autophagy could cooperate with apoptosis. Our results showed that MEHP caused LMP in EA.hy 926 cells after treatment with 200 μM MEHP for 12 h, but not 6 h, suggesting that LMP may have occurred after the activation

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of autophagic flux. Additionally, MEHP-induced LMP was attenuated when autophagy

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was inhibited. These findings indicated that the autophagic flux activation induced by MEHP can lead to LMP in EA.hy 926 cells. In earlier studies, inhibition of autophagy

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response by asparagine and wortmannin resulted in reduced LMP in resveratrol-treated cells (Hsu et al., 2009). Collectively, LMP induced by MEHP was autophagy dependent.

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In agreement with this, the upregulation of cathepsin B in cytosolic extracts, detected after treatment with 200 μM MEHP for 12 h, confirmed the MEHP-induced LMP in EA.hy 926 cells. Besides, the inhibition of autophagy resulted in reduced lysosomal release of cathepsin B in cells. Cathepsin B is an important mediator of apoptosis caused by the lysosomal-mitochondrial axis. Upon LMP, the cathepsins are released to the cytosol and mediated in part by Bid and Bax, subsequently triggering MOMP and

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cytochrome c release (Laforge et al., 2013). In our study, MOMP was induced in MEHP-treated EA.hy 926 cells as implied by the collapse of ΔΨm. As expected, the autophagy inhibition attenuated the MEHP-induced MOMP and the expression of

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cytochrome c in cells. Moreover, our results shown that cathepsin B inhibitor CA-074 Me efficiently repressed MEHP-induced cytochrome c release. Our data support that the release of cytochrome c was mediated by cathepsin B. These results indicated that

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MEHP-induced autophagy and the subsequent autophagy-dependent LMP are upstream and causative events for MOMP in MEHP-treated EA.hy 926 cells.

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Unleashed cytochrome c resulting from MOMP triggers the apoptosis (Mattiolo et al., 2015). As expected, MEHP induced apoptosis in EA.hy 926 cells after treatment for 12 h, and the inhibition of autophagy alleviated the MEHP-induced apoptosis. These results indicated that the apoptosis induced by MEHP was autophagy dependent. It was found

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previously that T-12 induced autophagy-dependent apoptosis in both in vitro and in vivo

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breast cancer models, and a new cyathane diterpene, cyathin Q exhibited anticancer activity via induction of autophagy-dependent apoptosis in colorectal cancer cells

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(Hamidullah et al., 2015; He et al., 2016) We found that CA-074 Me, the inhibitor cathepsin B attenuated MEHP-induced apoptosis. One plausible explanation for the

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observed MEHP-induced autophagy-dependent apoptosis in EA.hy 926 cells is that it is a final outcome of the progressive effect of MEHP. Briefly, it is an early MEHP-induced autophagy and a relatively later LMP in cells, and then LMP causes lysosomal release of cathepsin B and leads to MOMP, ultimately, triggering mitochondrial apoptosis. Both autophagy and apoptosis are well-controlled processes that play crucial roles in tissue homeostasis, development and disease. Increasing evidence supports that

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autophagy plays a protective role through inhibiting apoptosis, under normal or mild stressed conditions. For instance, activation of autophagy by epirubicin may protect cells from apoptosis in human breast cancer cells (Sui et al., 2013). Conversely, inactivation of

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autophagy may cause accumulation of abnormal proteins and organelles, thereby promoting apoptosis, under conditions of extreme stress. Here we found that the apoptosis was suppressed by inhibition of autophagy pathway, implicating that autophagy

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during pressure overload plays a detrimental role.

In summary, we suggest that MEHP-activated autophagic flux is an upstream event

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triggering LMP and succeeding apoptosis through the lysosomal-mitochondria axis (Figure 9). Since apoptosis is implicated in the pathogenesis of many different cardiovascular diseases, the inhibition of apoptosis holds promise as an effective therapeutic strategy (Nishida et al., 2008). Accordingly, either the inhibition of autophagy

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or the blockage of the crosstalk between autophagy and apoptosis, translate into a strong

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Conflict of Interest

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protection of human umbilical vein endothelial cells facing MEHP.

The authors have declared that there is no conflict of interest.

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Acknowledgments

This work was supported by the Liaoning Provincial Natural Science Foundation of

China (2014023004).

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Figure Legends Figure 1. Dose effects of MEHP on the cell viability of EA.hy 926 cells. EA.hy 926 cells were treated with increasing concentrations of MEHP (from 0 μM to 800 μM).

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After 24 h, cell viability was measured using MTT assays. All results are percentage of control, each bar represents mean ± SD (n=3). (** P < 0.01 vs. control).

Figure 2. MEHP induced LC3-Ⅱ formation from the early period of treatment.

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EA.hy 926 cells were treated with 200 μM MEHP for 3 h, 6 h, 12 h, and 24 h. (A) Western blot was performed on the cytoplasmic protein fraction of cells using

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antibodies against LC3 and β-actin. (B) Quantitative analyses of LC3-Ⅱ expressed in EA.hy 926 cells. Relative expression of LC3-Ⅱ was showed as a percentage of β-actin. Each bar represents mean ± SD (n=3). (** P < 0.01 vs. control). Figure 3. LMP induced by MEHP was autophagy dependent. (A) EA.hy 926 cells

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were treated with 200 μM MEHP for 6 h, 12 h, and 24 h. LMP was detected by AO

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staining and visualization by fluorescence microscopy (scale bar: 20 μm). (B) Quantitative analyses of AO staining in EA.hy 926 cells as described in (A). (C)

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EA.hy 926 cells were pretreated with 1 mM 3MA for 2 h, and then treated with 200

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μM MEHP for 12 h. LMP was detected by AO staining and visualization by fluorescence microscopy (scale bar: 20 μm). (D) Quantitative analyses of AO staining in EA.hy 926 cells as described in (C). Each bar represents mean ± SD (n=3). (** P < 0.01 vs. control; ## P < 0.01 vs. the group treated with MEHP).

Figure 4. Effect of 3MA on the expression of cathepsin B. Western blot was performed on the cytoplasmic protein fraction of cells using antibodies against cathepsin B and β-actin. Relative expression of cathepsin B was shown as a

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percentage of β-actin (n = 3). (A) EA.hy 926 cells were treated with 200 μM MEHP for 6 h, 12 h, and 24 h. (B) Quantitative analyses of cytosolic cathepsin B levels in EA.hy 926 cells as described in (A). (C) EA.hy 926 cells were pretreated with 1 mM

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3MA for 2 h, and then treated with 200 μM MEHP for 12 h. (D) Quantitative analyses of cytosolic cathepsin B levels in EA.hy 926 cells as described in (C). Each bar represents mean ± SD (** P < 0.01 vs. control; ## P < 0.01 vs. the group treated with

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MEHP).

Figure 5. Collapse of ΔΨm induced by MEHP was autophagy dependent. ΔΨm was

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analyzed in the EA.hy 926 cells after 200 μM MEHP treatment, using JC-1 staining, under a fluorescence microscopy (scale bar: 20 μm). ΔΨm was represented as the ratio of red fluorescence, corresponding to activated mitochondria, to green fluorescence, corresponding to less-activated mitochondria. (A) EA.hy 926 cells were

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treated with 200 μM MEHP for 6 h, 12 h, and 24 h. (B) Quantitative analyses of JC-1

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staining in EA.hy 926 cells as described in (A). (C) EA.hy 926 cells were pretreated with 1 mM 3MA for 2 h, and then treated with 200 μM MEHP for 12 h. (D)

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Quantitative analyses of JC-1 staining in EA.hy 926 cells as described in (C). Each bar represents mean ± SD (n=3). (** P < 0.01 vs. control;

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P < 0.01 vs. the group

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treated with MEHP).

Figure 6. Effect of 3MA on the expression of cytochrome c. Western blot was performed on the cytoplasmic protein fraction of cells using antibodies against cytochrome c and β-actin. Relative expression of cytochrome c was showed as a percentage of β-actin (n=3). (A) EA.hy 926 cells were treated with 200 μM MEHP for 6 h, 12 h, and 24 h. (B) Quantitative analyses of cytosolic cytochrome c levels in

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EA.hy 926 cells as described in (A). (C) EA.hy 926 cells were pretreated with 1 mM 3MA for 2 h, and then treated with 200 μM MEHP for 12 h. (D) Quantitative analyses of cytosolic cytochrome c levels in EA.hy 926 cells as described in (C). Each bar

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represents mean ± SD (** P < 0.01 vs. control; ## P < 0.01 vs. the group treated with MEHP).

Figure 7. Apoptosis Induced by MEHP Was Autophagy Dependent. (A) Apoptosis

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was assessed using TUNEL assay by fluorescence microscopy (scale bar: 20 μm) in EA.hy 926 cells treated with 200 μM MEHP for 6 h, 12 h, and 24 h. Green - TUNEL

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positive staining; Blue - DAPI counterstaining of nuclei. (B) Quantitative analyses of TUNLE positive cells as described in (A). Western blot was performed on the cytoplasmic protein fraction of cells using antibodies against caspase 3 and β-actin. Relative expression of caspase 3 was showed as a percentage of β-actin. (C) EA.hy

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926 cells were treated with 200 μM MEHP for 6 h, 12 h, and 24 h. (D) Quantitative

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analyses of cytosolic caspase 3 levels in EA.hy 926 cells as described in (C). (E) EA.hy 926 cells were pretreated with 1 mM 3MA for 2 h, and then treated with 200

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μM MEHP for 12 h. (F) Quantitative analyses of cytosolic caspase 3 levels in EA.hy 926 cells as described in (E). Each bar represents mean ± SD (n=3). (** P < 0.01 vs.

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control; ## P < 0.01 vs. the group treated with MEHP). Figure 8. Effects of CA-074 Me on the release of cytochrome c and apoptosis. EA.hy 926 cells were pretreated with 10 μM CA-074 Me for 2 h, and then treated with 200

μM MEHP for 12 h. (A) Western blot was performed on the cytoplasmic protein fraction of cells using antibodies against cytochrome c and β-actin. (B) Quantitative analyses of cytosolic cytochrome c levels in EA.hy 926 cells as described in (A). (C)

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Western blot was performed on the cytoplasmic protein fraction of cells using antibodies against caspase 3 and β-actin. (D) Quantitative analyses of cytosolic caspase 3 levels in EA.hy 926 cells as described in (C). Each bar represents mean ±

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SD (n=3). (** P < 0.01 vs. control; ## P < 0.01 vs. the group treated with MEHP).

Figure 9. The proposed pathway of MEHP-induced autophagy-dependent apoptosis in EA.hy 926 cells. MEHP-activated autophagy is an upstream event that may trigger

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LMP. LMP induced by MEHP causes lysosomal release of cathepsin B and leads to

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MOMP, ultimately, triggering mitochondrial apoptosis.

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Title: Mono(2-ethylhexyl) phthalate Induces Autophagy-Dependent Apoptosis

Highlights

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through Lysosomal-Mitochondrial Axis in Human Endothelial Cells

MEHP-induced apoptosis was autophagy-dependent.

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Lysosomal-mitochondrial axis was the main pathway.

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Autophagy triggered LMP, MOMP and apoptosis induced by MEHP.

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1.